Km and Vmax are constant for a given temperature and pH and are used to characterise enzymes. They can be used to identify types of inhibitors i. Vary the catechol concentration to find out Km. Start with your lowest catechol concentration and record the colour change every 30 seconds after adding the enzyme. Biologically, enzymes are essential molecules that speed up reactions in metabolic systems.
As a result, enzyme kinetics study the reaction rate of enzymes in various chemical settings. Many factors affect the speed of an enzyme.
The concentration of a substrate, temperature, inhibitors and pH influence the threshold of an enzyme in a chemical reaction.
With the help of linear relationships such as the Lineweaver-Burk plot, you can find the maximum rate of an enzyme. Begin by plotting the Michaelis-Menten equation to get a hyperbole curve. Then, use the reciprocal of the Michaelis-Menten equation to obtain a slope-intercept form of the enzyme activity. Before specific computer software, you would use graph paper to draw the line. Now, you use typical database software to plot the equation. So, knowing the initial rate, Vo, and the various concentration of the substrate, you can create a straight line.
An Example: The following concentrations of a methyltransferase were used in the SAM assay and the activites were calculated as per the protocol. Convert both columns of data in the above table to the inverse. This is 6. This assay is suitable for the simple and rapid estimation of protein concentration. The rate of formation of product now depends on the activity of the enzyme itself, and adding more substrate will not affect the rate of the reaction to any significant effect.
The rate of reaction when the enzyme is saturated with substrate is the maximum rate of reaction, Vmax. The relationship between rate of reaction and concentration of substrate depends on the affinity of the enzyme for its substrate.
This is usually expressed as the Km Michaelis constant of the enzyme, an inverse measure of affinity. For practical purposes, Km is the concentration of substrate which permits the enzyme to achieve half Vmax. An enzyme with a high Km has a low affinity for its substrate, and requires a greater concentration of substrate to achieve Vmax.
The Km of an enzyme, relative to the concentration of its substrate under normal conditions permits prediction of whether or not the rate of formation of product will be affected by the availability of substrate. An enzyme with a low Km relative to the physiological concentration of substrate, as shown above, is normally saturated with substrate, and will act at a more or less constant rate, regardless of variations in the concentration of substrate within the physiological range.
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